Material and methods
We used inbred lines described in Łukasiewicz et al. (2020), that were inbred via 4 generations of brother-sister mating and continued inbreeding for 6 more generations. In brief, mites collected from onion bulbs in Mosina, Wielkopolska, Poznań Poland, and inbred lines were initiated from the first generation of which hatched in the lab. The proportion of both male phenotypes used for crosses reflected that in the original population, giving rise 26 inbred lines founded by fighter males (fighter lines henceforth), and 15 inbred lines founded by scrambler males (scrambler lines). The consecutive generations were established by mating a randomly selected female with her sibling male of the same morph as the male founder. Choosing the same morph as the founder to establish each generation allowed us to maintain differences in morph proportions between F and S inbred lines throughout the inbreeding experiment (Fig. S1).
We established outbred populations enriched for scrambler or fighter genes (F and S populations henceforth) by mixing 12 inbred lines with the highest ratio of the desired morph after 10 generations of brother-sister mating described above. In this way we ensured that genes for the desired morph were also overrepresented among females. Furthermore, using inbred lines ensured effective population sizes of F and S populations were similar (i.e. contained about 24 genes randomly sampled from the original wild population), and that they carried much of the standing genetic variance present in the population collected from the field. We took six protonymphs (second juvenile stage) from each from inbred line (hence, there were 72 founders per population) and expanded them for two generations with random mating. The expanded populations were then subdivided into four F populations and four S populations by transferring 50 randomly selected protonymphs in a new vial. Next generation, each of these F and S populations was further subdivided it two treatments: gradual temperature increase (GTI, with three replicates per subpopulation) and control (one replicate). The GTI populations (FT and ST henceforth) were maintained at 25°C during first generation, with temperature increasing for the next two generations by 2°C, and then maintained at 29°C from third generation on (we did not increase the temperature further as our pilot study indicated that mites are unable to adapt to temperature over 30°C when the temperature increases 2°C per generation). Control populations (FC and SC henceforth) were maintained at 23°C at all generations. Each generation lasted 10 days, with c.a. three days needed for adult eclosion and 7 days for mating and reproduction (mating takes place immediately upon emergence and eggs are laid continuously starting ca. 24 h after mating). Each new generation was started by 50 randomly selected protonymphs. At each generation, we recorded the number of females, scrambler, and fighter males twice: immediately after adults eclosed and at the end of the reproduction period. A population was considered to be extinct if (i) the females did not lay any eggs, (ii) no larvae hatched from the laid eggs, or (iii) no adults of one or both sexes survived to adulthood.